The invention relates to the field of regeneration of cells, self-renewal of (micro)-organisms, the vegetative propagation of plant parts such as plant tissues or organs thereof, for example cells grown in tissue or organ culture, and more in particular to the seedless propagation of plants.
Renewal of plant and animal cells into more cells, tissues, organs and even whole plants and organisms is a process central to life that has been set to men's whims and desires already for a long time. Self-renewal of specific micro-organism starter cultures are used to ferment foods and drinks. Yet other cultures are useful for the metabolites they produce per se, such as produced by modern day's large scale fermentor cultures for the production of antibiotics or enzymes. Within the realm of animal cells, use of the renewed cultured cells, although being of fairly recent date, has taken great flight with the production of for example viral vaccines in cell- or tissue culture. Even more recent is the use of donor cells harvested from an individual, and grown and/or differentiated in culture, for transplantation purposes. Such cells (take for example bone marrow cells) are, after having been sufficiently regenerated and differentiated, proliferated or equipped with the desired characteristics, transplanted into a recipient for medical purposes. Shortly, such therapies will even include transgenic cells, transformed with modern recombinant techniques, that are thereby equipped with the desired characteristics and transplanted.
Regeneration is very well studied in plants, where it is crucial in vegetative propagation. In principle, plants can be propagated in two ways, via seeds or vegetatively without using seeds as starting material to obtain the desired plant. Both types of propagation may be impossible or undesirable under certain conditions. When propagation via seeds is unsatisfactory (when no seeds or too few of the desired seeds are formed or the desired seeds quickly loose their germination viability) then seedless propagation is often adopted. Also, when due to sexually crossing a very heterogenous progeny is or may be obtained due to its strong heterozygosity, propagation via seeds is often also considered unsatisfactory. Of course, seedless propagation of essentially seedless starting material may in a later phase give rise to the desired seeds, which can further be used to obtain the desired plants.
Within seedless propagation of plants two major fields can be distinguished: In vivo and in vitro vegetative propagation. In vivo vegetative propagation (via for example cuttings, splitting or division, layering, earthing up, grafting or budding, and other methods known to the gardener or horticulturist), has for many years played an important role in agriculture; e.g. with potatoes, apples, pears, many ornamental bulbs and tuberous plants like potatoes, many arboricultural crops, carnations, chrysanthemums, etc. Vegetative propagation is also very important in plant breeding: parent lines have to be maintained and propagated vegetatively for seed production; cloning is often required for setting up gene banks; adventitious shoot formation is needed to obtain solid mutants after mutation induction.
However, the classical methods of in vivo vegetative propagation often fall short (to slow, too difficult or too expensive) of that required or are completely impossible. In the last couple of decades, since the discovery that plants can be more rapidly cloned in vitro than in vivo, knowledge concerning vegetative propagation has grown quickly; this holds equally true for plants from temperate, subtropical as well as tropical regions. It has now even become possible to clone species by in vitro culture techniques that are impossible to clone in vivo. Different methods of in vitro vegetative or seedless propagation from plant starting material are for example using single-node cuttings, axillary branching, regeneration of adventitious organs (roots or shoots) on starting material such as explants or callus tissue and regeneration of plants from suspensions of, or even single, cells or protoplasts used as starting material. For the generation of transformed or transgenic plants, in vitro propagation is even considered a prerequisite, since it is the totipotency of individual plant cells that underlies most plant transformation systems.
To propagate plants from starting material in vitro, it is in principle necessary that at least one cell in the starting material is capable of regeneration. The ability to regenerate is for example determined by the genotype, the environmental conditions (nutrient supply, regulators and physical conditions) or the developmental stage of the plant, or combinations of these. It is well known that some families and genera have high regeneration ability: Solanacea (Solanum, Nicotiana, Petunia, Datura, and Lycopersion), Crucifera (Lunaria, Brassica, Arabidopsis), Generiaceae (Achimenes, Saintpaulia, Streptocarpus) Compositae (Chicornum, Lactuca, Chrysantemum), Liliaceae (Litium, Haworthia) Allium, Ornithogalum) but others, such as many decorative plants, woody species such as shrubs, conifers or trees, especially fruit trees, Rosacea, Alstroemeria, Euphorbia, and bulbs such as Tulipa, and others are notoriously difficult, even with in vitro techniques.
As indicated above, regeneration (self-renewal of (micro-)organisms and self-renewal of plants, animals or parts thereof, i.e. vegetative reproduction/propagation) can also be considered a repair strategy observed throughout the realm of micro-organisms, animal and plant species. Regeneration in plants for example comprises the formation of new tissues containing both root and shoot meristems, separate shoot or root meristems, plant organs or organ primordia from individual cells or groups of cells. Regeneration in general mimics the process of normal cellular and organ differentiation that takes place during plant development and results in the formation of the different plant organs. In normal development, early in ontogony, cells and tissues of common lineage diverge into often contrasting paths of development as they respond to developmental signals. This ability to develop in response to a specific signal is also known as cellular competence or cellular potentiality. As competent cells become committed to particular paths of differentiation, they are not readily diverted into other pathways; this restriction of the developmental potentiality of cells is referred to as determination.
Plant cells or groups of cells that under normal conditions are unable to initiate the formation of certain plant organs, meristems or organ primordia can often be stimulated by extracellular stimuli modifying the differentiation stage of the cell. Extracellular diffusible factors have shown to be essential for cellular redifferentiation in plant cells (Siegel and Verbeke, 1989 Science 244, 580-582). The perception of these signals at the cellular surface and the intracellular signal transduction that finally result in changes in transcriptional regulation provides cells with the ability to respond to such extracellular stimuli. Regeneration can result in the formation of either a shoot alone or a root alone or both together. Only after redifferentiation of a cell or tissue, regeneration is possible that results in differentiated tissue that again comprises the necessary three-dimensional layout of the emerging plant, the apical-basal or shoot-root body plan from which the mature desired plant can develop.
Indeed, central in in vitro techniques for seedless propagation are phytohormones and other factors often added to the culture medium that mimic these extracellular stimuli. For the process of regeneration of the original starting cell into a multicellular totipotent tissue underlying and preceding somatic embryogenesis or organogenesis in vitro in cell, tissue or explant cultures which lead to a fully differentiated plant again, in general a well balanced, and per plant species often different, phytohormone addition to the culture is required. Overall, a balance is required between auxins on the one hand and cytokinin on the other. After exogenous exposure to auxin (such as 2,4-dichlorophenoxyacetic acid (2,4-D), chloramben or dicamba) or cytokinin (such as 6-benzylaminopurine or zeatine) or both, cells or tissue react by development of the shoot-root body plan, for example by forming shoots and/or roots, sometimes readily, sometimes erratically especially when the proper balance between the hormones is not properly selected.
Regeneration in vitro and especially the manipulatable nature of in vitro culture thus depends mainly on the application of these two types of hormones, and also on the ability of the tissue to respond to phytohormonal changes during culture. In general, three phases of regeneration are recognisable. In the first phase, cells in the culture acquire “competence”, which is defined as the ability (not capacity) to respond to hormonal signals of organ induction. The process of acquisition of said organogenic competence is often referred to as “dedifferentiation” of differentiated cells to acquire organogenic competence. The competent cells in the culture are canalised and determined for specific tissue and organ formation for re-entry of quiescent cells into cell cycle, and organisation of cell division along the lines of the shoot-root body plan to form specific primordia and meristems under the influence of the phytohormone balance through the second phase. Especially auxin is thought to be involved in specific regenerative signal transduction pathways for adventitious root initiation, whereas cytokinin is thought to be involved in specific regenerative signal transduction pathways for adventitious shoot initiation.
Then the morphogenesis, the growing of the plant to its fully differentiated state, proceeds independently of the exogenously supplied hormones during the third phase.
Although the general principles governing regeneration via addition of exogenous phytohormones are thus fairly well understood, designing working in vitro culture protocols finding the right balance, the right time of administration or the right type or subtype of said hormones for a great many individual species is still more or less a process of trial-and-error. However, as already indicated above, for in vitro regeneration or seedless propagation of a great many plant species is a large interest, especially for those that are in general hard to propagate.
The invention provides a culture method for propagation of a plant from plant starting material wherein, especially in the phase of the development of the shoot-root body plan, root or shoot initiation is stimulated by introducing at least one recombinant gene product or functional fragment thereof in said starting material, for example by stimulating at least one signal transduction pathway for root or shoot initiation, said gene product or gene products for example derived from a gene or genes involved in the regulation of plant development, allowing reducing or omitting exogenous phytohormone addition to said culture in the regeneration process. In a preferred embodiment the invention provides a culture method for vegetative propagation of plants from plant starting material comprising regeneration of said starting material wherein during regeneration of said starting material at least one specific signal transduction pathway for adventitious root or shoot initiation is endogenously stimulated allowing reducing or omitting exogenous phytohormone addition to said culture, in particular wherein said pathway is endogenously stimulated by a recombinant gene product derived from a gene involved in the developmental regulation of regeneration, such as a gene or gene product involved in hormone production, a gene or gene product giving feed back on hormone production, or involved in the cascade of events leading to regeneration.
Preferably, the method as provided by the invention comprises at least one step of in vitro culture, since it is in in vitro culture that the auxins or cytokinins are most widely used, in the regeneration process, especially for plants that are notoriously difficult to regenerate for vegetative propagation such as many decorative plants, woody species such as shrubs, conifers or trees, especially fruit trees, Rosacea, Alstroemeria, Euphorbia, and bulbs such as Tulipa. However, clearly, said hormones are also commonly used in in vivo cultures as well, (in vivo cultures essentially being all crop or plant culture methods traditionally used in agriculture) where such hormones are commonly added by (root or stem) dipping, spraying or watering. Especially those plants that are propagated in an essential seedless way can now be regenerated or propagated more easily, consequently, in a preferred embodiment, the invention provides a culture method for essentially seedless propagation of plants from plant starting material comprising regeneration of said starting material wherein during regeneration at least one specific signal transduction pathway for adventitious root or shoot initiation endogenously is stimulated, e.g. by above mentioned gene product, allowing reducing or omitting exogenous phytohormone addition to said culture.